KEY FACTORS IDENTIFICATION OF
POLARIMETRIC METHOD FOR SPECIFIC
ROTATION DETERMINATION IN HONEY
Serrano, S.; Rodríguez, I.; Rincón, F.
Special collaboration: Jodral, M.
Department of Food Hygiene and
Technology
University of Cordoba, Spain
INTRODUCTION
Importance of specific rotation in honeys and honeydew honeys
(White, 1975)
Honeys and honeydew honeys have the property of rotating the
polarization plane of polarized light and this property depends largely on the
sugars of honey (their types and relative proportion).
The importance of this method is that appears to be valid to separate floral
honeys from honeydew honeys:
Floral honeys normally present higher content in fructose  Negative
Honeydew honeys present lower content in fructose but higher in
melezitose, erlose and glucose  Positive
INTRODUCTION
Importance of specific rotation in honeys and honeydew honeys
(White & Maher, 1953b)
Each carbohydrate has a specific angle of rotation of polarized light (specific
rotation) :
 Fructose  [α]D 20 = -92,4°
 Glucose  [α]D 20 = +52,7°
 Melezitose  [α]D 20 = +88,2°
 Erlose  [α]D 20 = +121,8°
INTRODUCTION
Importance of specific rotation in honeys and honeydew honeys
(White & Maher, 1953b)
Persano Oddo et al. (2000) propiose specific rotation ranges for several
Italian unifloral honeys :
 Citrus honey : -17 º to -9º
 Eucalyptus oney: -16º to -7º
 Heather honey: -15º to -13º
 Sunflower honey: -19º to -17º
 Thyme honey: -23º to -18º
 Fir honeydew honey: +8º to+19º
RANGES OVERLAPPING
INTRODUCTION
Importance of specific rotation in honeys and honeydew honeys
(White & Maher, 1953b)
Some special samples could be found:
 Floral honeys  Positive specific rotation
• Avocado honeys
• Multifloral honeys
Mixture??
 Honeydew honeys  Negative specific rotation
Mixture??
Adulterated ??
MORE RESEARCH IS NEEDED
OBJECTIVE
TO ANALIZE THE METHOD PROPOSED BY IHC (1999)
FOR SPECIFIC ROTATION DETERMINATION IN
HONEYS BY MEANS OF FRACTIONAL FACTORIAL
EXPERIMENTAL DESIGN WITH THE AIM OF
IDENTIFYING THE KEY PARAMETERS INFLUENCING
THE DIFERENTIATION BETWEEN BLOSSOM HONEYS
AND HONEYDEW HONEYS
SPECIFIC ROTATION METHOD (IHC, 1999)
1. SCOPE
The method can be applied to all honey samples. In particular, most of the honeydew
honeys have positive values of specific rotation whereas nectar honeys have negative
values.
2. DEFINITION
The specific optical rotation, [α]D 20 is the angle of rotation of polarised light at the
wavelength of the sodium D line at 20ºC of an aqueous solution of 1 dm depth and
containing 1g/ml of the substance.
3. PRINCIPLE
The angular rotation of a clear, filtered aeqous solution is measured by means of a
polarimeter.
The value is related to the carbohydrate composition. This procedure is based on
published methods (1,2)
SPECIFIC ROTATION METHOD (IHC, 1999)
4. REAGENTS
Carrez I solution. Dissolve 10.6 g potassium hexacyanoferrate(II), (K4Fe(CN)6 3H2O) in
distilled water and dilute to 100 ml.
Carrez II solution: dissolve 24 g zinc acetate (Zn(CH3COO)2⋅ 2H2O) in distilled water,
add 3 g of glacial acetic acid and dilute to 100 ml with distilled water.
5. EQUIPMENT
Polarimeter capable of angular rotation measurements accurate to within 0.05 circular
degrees, equipped with sodium lamp and 2-dm tube.
6. PROCEDURE
Sample preparation. If necessary, prepare the honey according to the section.
Determination.
Weigh 12 g honey (corresponding to about 10 g dry substance), dissolve it in distilled
water, add 10 ml of Carrez I solution and mix thoroughly for 30 seconds. Add 10 ml
Carrez II solution, mix again for 30 seconds and make up to volume in a 100 ml
volumetric flask with distilled water. The next day, filter the solution, rinse and fill a clean
2-dm polarimeter tube with the solution, place the tube in the polarimeter and read the
angular rotation (α). Measurements must be taken at a temperature of 20ºC.
SPECIFIC ROTATION METHOD (IHC, 1999)
7. CALCULATION AND EXPRESSION OF RESULTS
Specific angular rotation
[α]D 20 = α x 100 / l x p
where α = angular rotation found,
l = length in decimetres of the polarimeter tube
p = grams of dry matter taken
Results are at one decimal place
ANALYTICAL METHOD
STEP 1: Detection of apparent critical factors and their levels
affecting the specific rotation assay for honey samples
oPrevious experience
o Literature
“Brainstorming”
They were identified 11 apparent critical factors
ANALYTICAL METHOD
Symbol
ACF
Levels
Code
Actual
-1
+1
Honey type Source
X1
HT
blossom
honeydew
Liquefaction temperature (ºC)
X2
TL
30
40
Liquefaction time (min)
X3
TL
30
60
Prefiltering
X4
FI
yes
no
Sample weight (g) (1)
dilution volume of water (ml)
X5
X6
SWP
WV
low
25
high
75
Carrez I volume (ml)
X7
CIV
5
10
Carrez II volume (ml)
X8
CIIV
5
10
Final filtering (µ)
X9
FF
10-12
2-3
Polarimeter lenght tube (cm)
X10
PTL
10
20
Polaroimeter lecture system
X11
PLS
manual
automatic
ANALYTICAL METHOD
STEP 2: The evaluation of effects of each factor according to
considered responses. This step was carried out using a
fractional experimental design (Plackett-Burman):
o 32 experiments (runs) involving combinations of factors at
different levels : 2iv 11-6
oResponses considered were specific rotation (R1), pH of
lecture solution (R2) and conductivity of lecture solution (R3).
oHoney samples :
o OAK HONEYDEWHONEY
o MULTIFLORAL HONEY
ANALYTICAL METHOD
Polarimetric systems
Manual system
WXG - 4
Automatic system
ZUZI 412 AUTOMATIC
POLARIMETER
ANALYTICAL METHOD
RESULTS: Effects in terms of coded factors on each response en terms
specific rotation (R1), pH of lecture solution (R2) and conductivity of lecture solution
(R3). A strong effect was considered when p<0.20 and then the factor was
considered as influencing factor (IF).
R
Factors
X1
e
s
p
o
n
s
e
s
R1
R2
R3
14.72
-0.18
0.37
-0.14
-0.24
X2
X3
X4
X5
X6
0.12
X7
0.90
X8
-0.15
0.66
0.16
0.25
X9
X10
X11
8.97
R2
0.72
0.54
0.72
ANALYTICAL METHOD
DISCUSSION OF EFFECTS:
 Honey type (X1) on the specific optical rotation (R1) was 14,72, so mean
difference of specific optical rotation between both blossom (low level) and
honeydew (high level) honeys was 14,72º. This results are agree with other
reported previously, such as 19º (Dinkov, 2003)
Honey type (X1) on solution pH (R2) was -0.18, so for high level (honeydew)
the pH increases in 0.18 units
Honey type (X1) on solution conductivity (R3) was 0.37, so for high level
(honeydew) conductivity increases in 0.37 units
Prefiltering (X4) increases de pH of solution (R2=-0.14) and decreases the
electrical conductivity (R3=-0.24)
Dilution volume of water (X6) increases pH solution (R2=0.12)
ANALYTICAL METHOD
DISCUSSION OF EFFECTS:
 Carrez I Volumen (X7) increases the conductivity of solution (R3=0.90)
 Carrez II Volumen (X8) decreases the pH solution (R2=0.66)
 The polarimetric lecture system (X11) has a strong effect increasing
lectures 8.97 units respect the manual system when automatic system is
used
CONCLUSSIONS
Conclussions
1. Type of honey and polarimetric lecture system are
the key factors with strong influence on specific
rotation measure
2. Prefiltering, dilution volumen of water, and Carrez I
and Carrez II adition seem to be secondary influence
factors affecting to pH and conductivity of lecture
solution
3. Liquefaction temperature and time, sample weight,
final filtering and polarometric tube lenght have not
influence on specific rotation measure.
CONCLUSSIONS
Conclussions
4. Results of specific rotation in honeys require
same polarometric system to be compared.
the
5. IHC proposed method would be improved
if
specifications on
polarometric method and
requirement on not infuencing factors are eliminated or
adjusted with the aim to increase efectiveness and to
reduce cost